In cellular electrochemistry, ions respond to stimuli by constantly shuffling across cellular membranes to perform their physiological roles. This flow of ions, the electromotive force, leaves cells vulnerable to exogenous electromagnetic fields that can stimulate and/or modulate cellular activity. An irreparable link exists between changes in ionic concentration and the electric gradient of the cell (or its potential energy). Consequently, we can manipulate the physiology of the cell by altering its permeability to various ions, thereby modulating its electrical gradient. Only a few millivolts in excess of the resting membrane potential can stimulate a dramatic change in ion distribution within the cellular microenvironment. In excitable neural-type cells, electrical-stimulation-induced changes in membrane potential lead to the generation or inactivation of action potentials (AP). These AP trigger activities, such as nerve impulses in neurons or contraction in muscle cells. Within neural networks, targeted alteration of AP can prompt physiological changes that selectively stimulate or inactivate specific signals along nerve fibers. On the whole-organism level, electromagnetic fields applied directly to neural tissue, or transversely through the skull, produce profound effects that range from altered sensory perception to deviations in motor movement. Given this wealth of observable electromagnetic effects on neurological tissues, it is no surprise that other forms of electrical stimuli may elicit novel responses in an exposed biological system.

Figure 1. High speed spatiotemporal images of a NG108 neuroblastoma cell (left) before and (right) shortly after stimulation with a nanosecond pulsed electric field (nsPEF). A fluorescence signal was acquired using calcium green and was overlaid onto a differential interference contrast image.

Figure 2. Illustration of the hypothetical nsPEF-induced activation of protein kinase C (PKC) by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Translocation of fluorescent probes PLCδ-PH-EGFP (PH domain of phospholipase C bond to enhanced green fluorescent protein: green star) and GFP-C1-PKCγ(red star) demonstrate nsPEF-induced PIP2 hydrolysis. PLCδ-PH has a very high affinity for inositol trisphosphaste (IP3) and will move from the plasma membrane into the cytoplasm upon IP3 production following PIP2hydrolysis. C1 domain of PKCγ has a high affinity for diacylglycerol (DAG), and, therefore, the probe translocates from the cytoplasm to the membrane upon PIP2 hydrolysis (see Figure 3). Production of DAG and the release of intracellular calcium by binding IP3to endoplasmic reticulum receptors activates PKC, initiating many physiological processes.

Figure 3. Tracking of PIP2 hydrolysis using GFP-C1-PKCγ. CHO-K1 cells, expressing GFP-C1-PKCγand human muscarinic acetylcholine type1 (hM1) Gq/11-coupled receptor, exposed to oxotremorine (OxoM) (A,B,C, top left) and a single 600ns pulse (A, B, C, top right) showed similar response. GFP-C1-PKCγis a sensor that reports DAG production by translocation from the cytoplasm to the membrane upon rises in membrane DAG following PIP2 hydrolysis. Graphs D and E (left and right) display the cross section of the cell at 30 seconds post exposure and a temporal response of the cells before and after drug or nsEP stimulation, respectively.

Our research team is currently exploring the cellular response to high-amplitude, short-duration electrical pulses termed nanosecond pulsed electric fields (nsPEF). Seminal studies showed that nsPEF exposure can elicit changes in membrane potential, plasma membrane phospholipid scrambling, mitochondrial depolarization, calcium uptake, platelet aggregation, and, at intense or repeated exposures, cause cell death.1–7 Notably, these observations show no substantial uptake of propidium iodide, a common indicator of pore formation in the plasma membrane when electric pulses are applied for longer periods (μs to ms).8 Thus, we assume that nsPEF exposure causes the formation of small, ion-permeable pores, or nanopores, in the plasma membrane.2, 9,10 Unlike the larger pores, nanopores retain ion selectivity when exposed to electrical pulses, acting more like a channel, and persist for many minutes after only a single pulse exposure.9, 11 Most notably, the formation of nanopores in the plasma membrane elicits an acute and prolonged increase in intracellular calcium, an ion critical to many neurological and cellular processes.

We believe that nsPEF exposure is an ideal tool for the prolonged and non-invasive modulation of cell electrophysiology. Based on the hypothesis of nanopore formation, we investigated the dynamics of calcium entry into neuroblastoma cells. We used a highly sensitive electron multiplied CCD camera and precisely timed laser excitation to acquire high-resolution, spatiotemporal images of a single cell12 (see Figure 1). We visualized calcium entering from the sides nearest the electrodes in less than 1ms after perturbation by a single 600ns pulse, and filling the cell within 100ms. With extracellular calcium excluded from the bathing buffer, the intensity of the signal was reduced and the signal emanated from within the cell, suggesting calcium release from intracellular stores. By pre-exposing cells to the inhibitor thapsigargin in an effort to deplete intracellular calcium, we saw no change in signal, validating the intracellular origin of the signal. This finding was the first to definitively show, spatially, that nsPEF caused both extracellular uptake and intracellular release of calcium.

We hypothesize that the release of intracellular calcium is due, in part, to nsPEF-induced activation of intracellular pathways derived from the plasma membrane, namely the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) or PIP2. PIP2 is a well-characterized intracellular pathway that originates on the inner surface of the plasma membrane. It ultimately causes intracellular calcium release from the endoplasmic reticulum via inositol trisphosphate (IP3) receptors (see Figure 2), activating protein kinase C (PKC). To validate our hypothesis, we used a widely accepted optical probe of PIP2 hydrolysis and diacylglycerol (DAG) sensor GFP-C1-PKCγ (green fluorescent protein labeled C1 domain of protein kinase C): see Figure 3.13,14 We validated that a single nsPEF exposure can cause hydrolysis of PIP2, ultimately leading to increased DAG on the plasma membrane, and activation of PKC.

PKC triggers many physiological responses, including hormone secretion, AP propagation, and muscle contraction. Thus, by manipulating the electrochemistry of the cell with nsPEF, we can potentially elicit and control a number of biological responses. This single, exogenous, non-chemical stimulus can cause a prolonged activation of intracellular signaling cascades at a similar level to that of pharmaceutical treatment, but without the need for a specific cell surface receptor. The responses can last for minutes and can be delivered locally, precisely and without systemic drug administration. Electrical pulse delivery to cells offers scientists a new, instant, and simplified means of studying cellular physiology through direct, drug-free activation of cellular pathways. Non-invasive activation of PKC could be used to stimulate cognitive function or treat pain without pharmaceuticals or surgery. Future efforts will focus on validation of this effect in primary neuron cultures and evaluation of ion channels regulated by PIP2 hydrolysis.13

This study was supported by the Air Force Office of Scientific Research LRIR 13RH08COR.

Gleb P. Tolstykh, Gary L. Thompson

National Research Council

Fort Sam Houston, TX

Gleb Tolstykh holds a senior scientist associateship funded by the Air Force Office of Scientific Research.

Gary Thompson holds a postdoctoral scientist associateship funded by the Air Force Office of Scientific Research.

Hope T. Beier

Optical Radiation BranchAir Force Research Laboratory

Fort Sam Houston, TX

Hope T. Beier is a research biomedical engineer in the 711 Human Performance Wing.